Predicting histologic differentiation of solitary hepatocellular carcinoma up to 5 cm on gadoxetate disodium-enhanced MRI

Background To establish a preoperative score based on gadoxetate disodium-enhanced magnetic resonance imaging (EOB-MRI) and clinical indicators for predicting histologic differentiation of solitary HCC up to 5 cm. Methods From July 2015 to January 2022, consecutive patients with surgically proven solitary HCC measuring ≤ 5 cm at preoperative EOB-MRI were retrospectively enrolled. All MR images were independently evaluated by two radiologists who were blinded to all clinical and pathologic information. Univariate and multivariate logistic regression analyses were performed to identify significant clinicoradiological features associated with poorly differentiated (PD) HCC, which were then incorporated into the predictive score. The predictive score was validated in an independent validation set by area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy. Results A total of 182 patients were included, 42 (23%) with PD HCC. According to the multivariate analysis, marked hepatobiliary phase hypointensity (odds ratio [OR], 9.98), LR-M category (OR, 5.60), and serum alpha-fetoprotein (AFP) level > 400 ng/mL (OR, 3.58) were incorporated into the predictive model; the predictive score achieved an AUC of 0.802 and 0.830 on the training and validation sets, respectively. The sensitivity, specificity, and accuracy of the predictive score were 66.7%, 85.7%, and 81.3%, respectively, on the training set and 66.7%, 81.0%, and 77.8%, respectively, on the validation set. Conclusion The proposed score integrating two EOB-MRI features and AFP level can accurately predict PD HCC in the preoperative setting. Supplementary Information The online version contains supplementary material available at 10.1186/s13244-022-01354-w.

Keywords Carcinoma (Hepatocellular), Histologic differentiation, Gadolinium ethoxybenzyl DTPA, Magnetic resonance imaging Background Primary liver cancer is the sixth most common malignancy and the fourth leading cause of cancer-related death globally, and hepatocellular carcinoma (HCC) accounts for around 75-85% of these cases [1]. Liver resection constitutes the backbone for curative treatment of HCC in patients with preserved liver function. However, its application is dampened by high incidence of postoperative recurrence that approaches 60-70% at 5 years [2].
Histologic differentiation is an established prognostic indicator in HCC. In specific, poorly differentiated HCC (PD HCC) has been associated with worse overall survival and/or disease-free survival after liver resection [3], liver transplantation [4], and radiofrequency ablation [5]. Therefore, accurate assessment of tumor differentiation, particularly in the preoperative stage, can help tailor individualized treatment decision-making and improve postoperative survival. Nevertheless, the degree of differentiation is accessible only through histologic examination following invasive biopsy or surgery, which is prone to sampling errors or can only be evaluated postoperatively.
Fortunately, imaging techniques can provide important information regarding tumor biology and heterogeneity in a noninvasive manner. For degree of differentiation, studies have shown that tumor signal intensity in the hepatobiliary phase (HBP) of gadoxetate disodium-enhanced magnetic resonance imaging (EOB-MRI) was associated with histologic grade of HCC [6][7][8][9]. Other imaging features, including the "washout" appearance [10], lower tumor-to-liver enhancement ratio in early arterial phase, absence of "capsule, " arterial phase peritumoral hyperenhancement [11], and peritumoral hypointensity in HBP, were also reported to correlate with moderately differentiated (MD) or PD HCC [9].
Although results so far have been promising, the evidence qualities of previous studies were limited by potentially biased radiology-pathology correlation, as most of these studies did not pose strict restrictions on tumor number and size [7,9,11]. Nevertheless, considering the markedly heterogenous nature of HCC (especially in multifocal and/or large tumors) [12,13], sampling errors may have greatly impacted the results. For example, multifocal HCCs may have distinct degrees of differentiation among tumors, but most prior work only evaluated the dominant tumors. As for first-line curative-intent treatment options, solitary HCC ≤ 5 cm was within the Milan criteria, by considering possible influence of tumor differentiation on prognosis, it may help prompting tailored treatment selection among transplant, locoregional treatment, and resection. Therefore, the imaging indicators of tumor differentiation in solitary HCC ≤ 5 cm remain to be defined.
Thus, this study aimed to establish an easy-to-use score based on preoperative EOB-MRI and clinical indicators for predicting histologic differentiation of solitary HCC up to 5 cm.

Materials and methods
This single-center retrospective study was approved by the institutional review boards at West China Hospital, Sichuan University, and the requirements to obtain written informed consent were waived.

Patients
From July 2015 to January 2022, consecutive patients were enrolled according to the following inclusion criteria: (1) age ≥ 18 years; (2) with history of chronic hepatitis B viral infection and/or liver cirrhosis not attributable to congenital or vascular abnormalities; (3) undergoing EOB-MRI within 1 month prior to liver resection; (4) with surgically proven single HCC measuring ≤ 5 cm at preoperative EOB-MRI; and (5) with complete baseline clinical information (detailed below) within 2 weeks before surgery.
The exclusion criteria were as follows: (1) any previous treatment for HCC; (2) the postoperative pathology report was inadequate for determining tumor differentiation; and (3) the MR images were of insufficient quality (e.g., severe motion artifact) for image analysis.
Baseline preoperative clinical information, including patient demographics, underlying liver diseases, presence or absence of cirrhosis, Child-Pugh classes, and key laboratory data (e.g., alpha-fetoprotein [

Image acquisition
The EOB-MR images were acquired on various 1.5 T or 3.0 T MR systems. The MRI sequences included: T2-weighted imaging (T2WI); diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) maps; in-and opposed-phase T1-weighted imaging; and dynamic T1-weighted imaging before and after injection of gadoxetate disodium (Xianai ® , Zhengdatianqing Pharmaceutical Group; Primovist ® , Bayer Schering Pharma AG) in the late arterial phase (AP), portal venous phase (PVP), transitional phase (TP), and HBP. Detailed imaging acquisition parameters are shown in Additional file 1: A1 and Table S1.

Image analysis
All de-identified MR images were independently reviewed by two fellowship-trained radiologists (with 5 and 7 years of experiences in liver MRI, respectively). The reviewers were aware that all patients had HCC but were blinded to all clinical, laboratory, and pathologic results. All disagreements between the two reviewers were resolved by a senior fellowship-trained radiologist who had over 20 years of experience in liver MRI.
The reviewers independently evaluated all the lesions regarding the presence/absence/degree of imaging features which had been reported to correlate with the degree of tumor differentiation, tumor burden, and HCC aggressiveness, including: (1) imaging features correlated with tumor differentiation (e.g., HBP signal intensity, degree of diffusion restriction); (2) all Liver Imaging Reporting and Data System (LI-RADS) version 2018 major and ancillary features (except for those related to growth and ultrasound visibility), LR-M features, TIV features, and LI-RADS category [14]; (3) imaging features profiling peritumoral changes (e.g., peritumoral HBP hypointensity [15], peritumoral T2WI hyperintensity, peritumoral biliary ductal dilation); and (4) other reported prognostic imaging features (e.g., intratumoral artery, non-smooth tumor margin, complete "capsule, " tumor growth pattern). The definitions and illustrations of the imaging features are detailed in Additional file 1: Table S2.

Histopathology
Information on tumor differentiation was extracted from routine pathological reports as the reference standard for tumor differentiation, which was recorded by another radiologist without knowing patient's imaging and clinical data. Based on the standard operation procedure of our institution, a 7-site sampling procedure was performed to ensure adequate and reliable assessment of tumor differentiation. In specific, one piece of tissue was each sampled at the transition area between tumor and surrounding liver tissues at a ratio of 1:1 at 12, 3, 6 and 9 o'clock from the less bleeding and necrotic areas. One piece was sampled within the tumor area free from bleeding and necrosis, and one piece each was sampled, respectively, from proximal (≤ 1 cm to the tumor) and distant liver parenchyma (> 1 cm to the tumor). Tumor differentiation was determined according to the World Health Organization (WHO) criteria [16]. The higher pathological grade was recorded when the evaluated HCC had mixed tumor grades.

Statistical analysis
Continuous variables were presented as mean ± standard deviation or median (interquartile range [IQR]) and analyzed with either the Student's t test or the Mann-Whitney U test, whereas categorical variables were presented as the numbers of cases (percentages) and analyzed with either the Chi-square test or the Fisher 's exact test, where applicable.
Interobserver agreement between the two radiologists was assessed by calculating the intraclass correlation coefficient (ICC) for continuous imaging features, the Cohen's κ value for binary imaging features, and the weighted κ value for categorical/ordinal imaging features, respectively.

Development of the predictive score for tumor differentiation
All eligible patients were randomly assigned into a training set and an independent validation set at a ratio of 7:3, while guaranteeing that the distributions of poorly and non-poorly differentiated HCCs between two sets were comparable.
In the training dataset, significant clinical and imaging predictors of PD HCC were selected by univariate logistic regression analysis. Afterward, controlling for patient age, gender, and underlying liver cirrhosis, all predictors with p < 0.1 at univariate logistic regression analysis were fit into a multivariate logistic regression model with backward stepwise method based on fivefold cross-validation, and the best-fit feature combination was obtained using the Akaike Information Criterion. To improve clinical utility and model simplicity, continuous variables were converted to categorical or binary ones according to normal range or clinical relevance. Inter-variable correlations were estimated by the pairwise Spearman's correlation analysis; when significant collinearity was observed, variables with the largest odds ratio (OR) at univariate logistic regression analysis were kept for further analysis. An HCC differentiation score was developed based on the significant predictors at multivariate logistic regression analysis weighted by their β regression coefficients, with the largest β coefficient scaled as 10 points. The Youden's index was used to analyze the receiver operating characteristic curve to determine the optimal threshold of the HCC differentiation score for predicting PD HCC.

Validation of the predictive score for tumor differentiation
The discriminative accuracy of the HCC differentiation score was evaluated with area under the receiver operating characteristic curve (AUC), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. Model calibration was assessed by the calibration curve with the Hosmer-Lemeshow test. Decision curve analysis was performed to investigate the clinical usefulness of the HCC differentiation score by quantifying the net benefits at different threshold probabilities.
The R software (version 3.5.1) and SPSS (version 26) were used to perform the statistical analysis. Two-tailed p ≤ 0.05 was considered statistically significant.

Patient characteristics
A total of 182 patients (mean age ± standard deviation, 52.9 ± 11.2; range, 28-75; 145 men) were included in this study, among whom 128 and 54 patients were divided into the training and validation sets, respectively ( Fig. 1).
Baseline clinical characteristics are presented in Table 1; frequencies of EOB-MRI features are summarized in Table 2; comparisons in baseline clinical characteristics of patients between the training and validation sets are shown in the Additional file 1: Table S3, and interobserver agreement for all imaging features and LI-RADS categories are detailed in Additional file 1: Table S4.
On the training set, PD HCC was pathologically confirmed in 30 patients (23.4%; 30/128). No significant differences in baseline clinical parameters were observed between patients with PD HCC and those with well differentiated (WD) or MD HCC (p = 0.103-1.000), except for serum AFP level (p = 0.010). Moreover, rim arterial phase hyperenhancement, marked diffusion restriction, LR-M category and marked HBP hypointensity were significantly more frequent in PD HCC than in WD/MD HCC. Other EOB-MRI features were similar in both groups (p = 0.071-1.000).
On the validation set, PD HCC was pathologically confirmed in 12 patients (22.2%; 12/54). There were no significant differences in baseline clinical variables between patients with PD HCC and those with WD/ MD HCC (p = 0.058-1.000), except for PT (p = 0.012). Additionally, the frequency of marked HBP hypointensity was significantly higher in PD HCC compared with WD/MD HCC. Other EOB-MRI features were not significantly different in both groups (p = 0.222-1.000).
Elevated serum carcinoembryonic antigen (> 5 ng/ mL) was more frequently observed for patients in the training dataset (7.8% vs. 0%, p = 0.035) than those in the validation dataset. No difference in other baseline    clinical characteristics was detected between the training and testing dataset (p = 0.134-0.784).  Fig. 2. The total score was calculated by adding the individual points of each variable, ranging from 0 to 23 points. According to Youden's index, the optimal threshold for predicting PD HCC was 6.5 points. Typical patients with PD HCC or MD HCC are shown in Fig. 3.

Score assessment and validation
On the training set, the HCC differentiation score exhibited an AUC of 0.802 (0.703-0.900) with sensitivity, specificity, PPV, NPV, and accuracy of 66.7%, 85.7%, 58.8%, 89.4%, 81.3%, respectively. Calibration plot demonstrated a good consistency between the score-predicted probabilities and the actual PD HCC estimates on the training set (p = 0.883; Additional file 1: Fig. S1a). Decision curves revealed that the predictive score provided a larger net benefit than that of assuming all patients had PD HCC when the threshold probability was greater than 0.1 on the training set (Additional file 1: Fig. S2a).
On the validation set, the predictive score yielded an AUC of 0.830 (0.693-0.966) with sensitivity, specificity, PPV, NPV and accuracy of 66.7%, 81.0%, 50.0%, 89.5%, 77.8%, respectively (Table 4). Calibration plot showed that the score-predicted probabilities were closely consistent with the actual PD HCC estimates on the validation set (p = 0.963; Additional file 1: Fig. S1b). In terms of the clinical utility, the predictive score exhibited a larger net benefit than that of assuming all patients had PD HCC when the threshold probability was greater than 0.08 on the validation set (Additional file 1: Fig. S2b).

Discussion
In patients with surgically-confirmed solitary HCC ≤ 5 cm, we found that two EOB-MR imaging features (marked hepatobiliary phase hypointensity and the LR-M category) along with serum AFP > 400 ng/mL were significantly associated with poor tumor differentiation. Based on these indicators, we developed and validated an easy-to-use scoring system which allowed accurate preoperative assessment of tumor differentiation.
Marked HBP hypointensity, defined as tumor signal intensity in the HBP lower than that of liver and similar to or lower than that of intrahepatic vessels, was present in 43 (24%) patients and significantly associated with poor tumor differentiation. This finding was in line with previous studies, in which lower tumor signal intensity in the HBP was associated with worse differentiation [7,8]. One possible explanation for this outcome is that the expressions of organic anion transporters on the cell membrane, which are responsible for the uptake of gadoxetic disodium and signal intensity in the HBP, decrease gradually as the tumor de-differentiates [17]. However, the assessments of HBP signal intensity were mostly performed via quantitative analyses based on manual regions of interest placement in prior works [7,8]. Despite allowing accurate quantifications of signal intensities, this approach may suffer from suboptimal generalizability, as signal intensity in the HBP is largely affected by vendor and acquisition parameter variations. In contrast, using intrahepatic vessels as a reference, we explored a reproducible and effective semi-quantitative way of measuring relative tumor signal intensity on HBP images, which could aid in preoperative evaluation of HCC differentiation.
The LR-M category is defined as lesions that are probably or definitely malignant but not HCC specific. Any targetoid appearances or nontargetoid mass with one or more features (including infiltrative appearance, marked diffusion restriction, necrosis or severe ischemia, and other feature that suggests non-HCC malignant), were considered to be sufficient for LR-M categorization [14]. And it was another imaging indicator of PD HCC. Our results were in consistent with the study of Shin et al., who found that the LR-M HCCs had poorer histologic differentiation than those LR-4/5 tumors [18]. In addition to worse differentiation, the LR-M category has also been reported to correlate with other aggressive pathomolecular characteristics of HCC, including increased stemness features (i.e., expression of CK19) [19], more pronounced macrotrabecular pattern, more frequent microvascular invasion and sinusoid-like microvascular pattern, and a     more hypoxic and fibrotic tumor microenvironment [20]. As a result, LR-M category was widely-proved as an independent risk factor for worse prognosis in HCC [18,21]. Serum AFP level > 400 ng/mL was the only laboratory indicator significantly associated with poor tumor differentiation in our study, which was in line with existing evidence. As the most established biomarker in HCC, higher serum AFP levels have been associated with increased tumor aggressiveness (e.g., worse tumor differentiation [22], increased incidence of microvascular invasion [23], macrotrabecular-massive subtype [20,24], proliferative HCC [25]), and worse posttreatment prognosis [26][27][28]. Our work further confirmed its utility, alone and in combination with other imaging features, in identifying PD HCC.
Our findings had important clinical implications. Methodologically, considering marked intra-and intertumoral heterogeneity in HCC, satisfactory radiologypathology correlation was achieved via the rigorous multi-site sampling method as we only enrolled patients with solitary tumors measuring ≤ 5 cm. Of note, identification of poorly-differentiated elements in small tumors might have been more challenging compared with tumors > 5 cm since these components are less frequently found in smaller HCCs [29]. However, our model still demonstrated AUCs over 0.80 on both the training and testing datasets. Moreover, all three indicators included in the final scoring system were identified as significantly associated with PD HCC in three out of the five folds of cross-validation during modeling. These outcomes